Backgrounds and Objectives: Mycoplasmas are distinguished phenotypically from other bacteria by their minute size and total lack of a cell wall and are involved in urogenital infections in both male and females. The aim of this study was to evaluate the presence of Mycoplasma genus and Mycoplasmahomonis and genitalium in urine samples of athletes and non-athletes.Methods: In this study, urine samples were taken from 50 athlete and non-athlete males and were transported to laboratory. After DNA extraction, PCR was performed using specific primers to identify Mycoplasma genus and species hominis and genitalium.Results: Among 100 samples, 5 were positive for Mycoplasma genus which all the 5 isolate belonged to non-athlete persons. In 5 positive samples, 3 belonged to Mycoplasmahominis and 2 belonged to Mycoplasmagenitalium.Conclusions: This study showed the presence of Mycoplasma in athletes were less than non-athletes. In addition PCR was a sensitive and precise method to identify Mycoplasma genus and species.